Methods To Diagnose Treat and Prevent Bone Loss

ABSTRACT

Methods of diagnosing and preventing bone loss and/or enhancing bone formation are disclosed. The invention additionally provides methods of diagnosing a predisposition to bone loss. The methods mathematically combine the information provided by imaging tests with the information provided by biomarkers to provide an index value. The index value is used for diagnosis of bone diseases, and to assess the progress of treatment of bone diseases.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 10/157,745, entitled“Methods to Diagnose Treat and Prevent Bone Loss,” filed May 28, 2002,the disclosure of which is incorporated herein, in its entirety byreference.

U.S. Ser. No. 10/157,745, in turn, is related to U.S. Provisional PatentApplication Ser. No. 60/293,898, and U.S. Provisional Patent ApplicationSer. No. 60/293,489, both filed on May 25, 2001, from which priority isclaimed under 35 USC §119(e)(1), and which applications are incorporatedherein by reference in their entireties.

TECHNICAL FIELD

The invention relates generally to methods for diagnosing, screening,prognosing, and treating diseases. More particularly, the presentinvention relates to a method for diagnosing, screening or prognosingchanges in bone loss, bone architecture or bone formation in humans oranimals, and for determining the severity and cause of the disease bymathematically combining morphological data with metabolic data obtainedusing biomarkers.

BACKGROUND

Osteoporosis is a major public health issue caused by a reduction inbone mineral density in mature bone and results in fractures afterminimal trauma. The most common fractures occur in the vertebrae, distalradius (Colles' fracture) and hip. An estimated one-third of the femalepopulation over age 65 will have vertebral fractures, caused in part byosteoporosis. Moreover, hip fractures are likely to occur in about onein every three woman and one in every six men by extreme old age.

Two distinct phases of bone loss have been identified. One is a slow,age-related process that occurs in both genders and begins at about age35. This phase has a similar rate in both genders and results in lossesof similar amounts of cortical and cancellous bone. Cortical bonepredominates in the appendicular skeleton while cancellous bone isconcentrated in the axial skeleton, particularly the vertebrae, as wellas in the ends of long bones. Osteoporosis caused by age-related boneloss is known as Type II osteoporosis.

The other type of bone loss is accelerated, seen in postmenopausal womenand is caused by estrogen deficiency. This phase results in adisproportionate loss of cancellous bone, particularly trabecular bone.Osteoporosis due to estrogen depletion is known as Type I osteoporosis.The main clinical manifestations of Type I osteoporosis are vertebral,hip and Colles' fractures. The skeletal sites of these manifestationsboth contain large amounts of trabecular bone. Bone turnover is usuallyhigh in Type I osteoporosis. Bone resorption is increased but there isinadequate compensatory bone formation. Osteoporosis has also beenrelated to corticosteroid use, immobilization or extended bed rest,alcoholism, diabetes, gonadotoxic chemotherapy, hyperprolactinemia,anorexia nervosa, primary and secondary amenorrhea, transplantimmunosuppression, and oophorectomy.

The mechanism by which bone is lost in osteoporotics is believed toinvolve an imbalance in the process by which the skeleton renews itself.This process has been termed bone remodeling. It occurs in a series ofdiscrete pockets of activity. These pockets appear spontaneously withinthe bone matrix on a given bone surface as a site of bone resorption.Osteoclasts (bone dissolving or resorbing cells) are responsible for theresorption of a portion of bone of generally constant dimension. Thisresorption process is followed by the appearance of osteoblasts (boneforming cells) that then refill with new bone the cavity left by theosteoclasts.

In a healthy adult subject, osteoclasts and osteoblasts function so thatbone formation and bone resorption are in balance. However, inosteoporotics an imbalance in the bone remodeling process develops whichresults in bone being replaced at a slower rate than it is being lost.Although this imbalance occurs to some extent in most individuals asthey age, it is much more severe and occurs at a younger age inpostmenopausal osteoporotics, following oophorectomy, or in iatrogenicsituations such as those resulting from the use of corticosteroids orimmuno suppressors.

The diagnosis and management of bone related disease, such asosteoporosis, typically requires information about bone turnover andbone mass. Determinations of bone turnover have historically beenperformed utilizing standard serum, urine and/or sweat laboratory testsincluding fasting calcium/creatinine, hydroxyproline, alkalinephosphatase and/or osteocalcin/bone growth protein utilizing standardhigh pressure liquid chromatography (HPLC) techniques. To illustrate,whenever bone formation occurs (calcium deposition) or bone resorptionoccurs (calcium breakdown), various chemical reactions occur within thebody that elevate the presence of certain indicators in the blood andurine suggesting changes in the calcium/bone mineral status. Biomarkers,however, typically lack information on the severity or stage of adisease and, additionally, on the morphological condition of an organ ortissue.

Recently, several new bone specific assays have been developed whichenable bone turnover to be evaluated with an ELISA/EMIT immunoassayformat. Descriptions of these immunoassay formats can be found in U.S.Pat. Nos. 5,973,666, 5,320,970, 5,300,434 and 5,140,103. The labelingfor the new assays utilize a biochemical marker to quantify boneresorption and/or formation and provides information on bone turnover.

For diagnosis of bone diseases, U.S. Pat. No. 6,210,902 describesdetecting collagen breakdown products in serum or urine by using two ormore immunoassays, and forming a ratio between the concentration of onefragment and a second fragment to form an index to determine the rate ofbone resorption. In another method of forming an index of biomarkerresults, U.S. Pat. No. 5,962,236 obtains a ratio of free lysylpyridinoline cross-links and creatinine content to form a urinary indexof bone resorption to diagnose bone disease. Further, the use of two ormore biomarkers to diagnose a disease, where a neural network is firsttrained and the trained neural network is then used to analyze theexperimental data to produce a diagnostic value is disclosed in U.S.Pat. Nos. 6,306,087 and 6,248,063.

Bone mass determinations, on the other hand, have been traditionallyperformed by using various x-ray based techniques including single anddual-photon absorptiometry (SPA and DPA), quantitative computedtomography (QCT), and dual-energy absorptiometry (DXA). Imaging testssuch as x-rays, ultrasound, computed tomography and MRI can providedetailed information about the morphological condition of an organ or atissue and on the severity or the stage of a disease process. However,such imaging techniques typically lack information on the metabolicactivity of various tissues and organs and, in diseased states, cannotgive an estimate of the rate of progression or the prognosis of adisease.

U.S. Pat. No. 5,785,041 describes a computer system for displayingbiochemical data with data from densitometric bone measurement todetermine whether bone formation or bone resorption is occurring.

Thus, there remains a need for methods and devices for diagnosing,prognosticating and monitoring of osteoporosis by combining theinformation provided by imaging tests with the information provided bybiomarkers.

SUMMARY

The present invention provides novel methods for the diagnosis ofdiseases, particularly bone related diseases, where using a combinationof several independent measurements or tests provides for greaterdiagnostic power.

In one aspect, the invention includes using a mathematical function torelate the level of one or more biomarkers with a numerical valuerelating to one or more imaging descriptors comprising predeterminedfeatures from images defining bone disease characteristics to obtain atest value, and comparing the test value with a control value, wherein atest value which differs from the control value by a predeterminedamount is indicative of bone disease.

These and other aspects of the present invention will become evidentupon reference to the following detailed description and attacheddrawings. In addition, various references are set forth herein whichdescribe in more detail certain procedures or compositions, and aretherefore incorporated by reference in their entirety.

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of protein chemistry, biochemistry,recombinant DNA techniques and pharmacology, within the skill of theart. Such techniques are explained fully in the literature. See, e.g.,T. E. Creighton, Proteins: Structures and Molecular Properties (W. H.Freeman and Company, 1993); A. L. Lehninger, Biochemistry (WorthPublishers, Inc., current addition); Sambrook, et al., MolecularCloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology(S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington'sPharmaceutical Sciences, 18th Edition (Easton, Pa: Mack PublishingCompany, 1990); Carey and Sundberg Advanced Organic Chemistry 3.sup.rdEd. (Plenum Press) Vols A and B (1992).

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.

I. Definitions

In describing the present invention, the following terms will beemployed, and are intended to be defined as indicated below.

By “bone loss” is meant an imbalance in the ratio of bone formation tobone resorption resulting in less bone than desirable in a patient. Boneloss may result from osteoporosis, bone fractures, osteotomy,periodontitis, or prosthetic ingrowth. Bone loss may also result fromsecondary osteoporosis that includes glucocorticoid-inducedosteoporosis, hyperthyroidism-induced osteoporosis,immobilization-induced osteoporosis, heparin-induced osteoporosis orimmunosuppressive-induced osteoporosis. Bone loss can be monitored, forexample, using bone mineral density measurements described below.

By “increased bone accretion” is meant that bone accumulation in asubject. Such increased bone accretion is determined herein by measuringbone mineral density (BMD). For example, bone accretion can bedetermined using an animal model, such as an ovariectomized mouse, dogand the like. The animal is administered the test compound and bonemineral density (BMD) measured in bones that are normally depleted inType I or Type II osteoporosis, such as bones of the appendicular and/oraxial skeleton, particularly the spine including the vertebrae, as wellas in the ends of long bones, such as the femur, midradius and distalradius. Several methods for determining BMD are known in the art. Forexample, BMD measurements may be done using, e.g., dual energy x-rayabsorptiometry or quantitative computed tomography, and the like. (See,the examples.) Similarly, increased bone formation can be determinedusing methods well known in the art. For example, dynamic measurementsof bone formation rate (BFR) can be performed on tetracycline labeledcancellous bone from the lumbar spine and distal femur metaphysics usingquantitative digitized morphometry (see, e.g., Ling et al.,Endocrinology (1999) 140:5780-5788. Alternatively, bone formationmarkers, such as alkaline phosphatase activity (see, e.g., Farley etal., Calcif. Tissue Int. (1992) 50:67-73) and serum osteocalcin levels(see, e.g., Taylor et al., Metabolism (1988) 37:872-877 and Baylink etal., 10th Annual Congress of Endocrinology, San Francisco, Calif. (1996)Abstract P1-945), can be assessed to indirectly determine whetherincreased bone formation has occurred.

As used herein, the terms “treat” or “treatment” are usedinterchangeably and are meant to indicate a postponement of developmentof bone loss symptoms and/or a reduction in the severity of suchsymptoms that will or are expected to develop. The terms further includeameliorating existing bone or cartilage deficit symptoms, preventingadditional symptoms, ameliorating or preventing the underlying metaboliccauses of symptoms, and/or encouraging bone growth.

As used herein, the term “subject” encompasses mammals and non-mammals.Examples of mammals include, but are not limited to, any member of theMammalia class: humans, non-human primates such as chimpanzees, andother apes and monkey species; farm animals such as cattle, horses,sheep, goats, swine; domestic animals such as rabbits, dogs, and cats;laboratory animals including rodents, such as rats, mice and guineapigs, and the like. Examples of non-mammals include, but are not limitedto, birds, fish and the like. The term does not denote a particular ageor gender.

The term “biomarker” encompasses any molecule having altered (e.g.,reduced or elevated) levels when a particular disease or condition in asubject is present as compared a normal (non-diseased) subject.Non-limiting examples of suitable biomarkers include polynucleotides(e.g., genes, a piece of DNA, a piece of RNA, an oligonucleotide);polypeptides (e.g., enzymes, etc.); lipids; a component of a membrane; acomponent of an organelle; oligo- or polysaccharides; metals; and/or anelement that is naturally occurring in a mammal including a human in aphysiologic or a diseased state. The presence, absence and/or amount ofa biomarker in a tissue or a bodily fluid can be indicative of adiseased state in a tissue or can be used to assess the metabolicactivity of normal or diseased tissue or can be used to prognosticatedisease state. Alterations in the concentration of a biomarker in atissue or a bodily fluid, e.g., an increase or a decrease above or belowthe normal range expected under physiologic conditions in said tissue orbodily fluid, can be indicative of a diseased state in a tissue or canbe used to assess the metabolic activity of normal or diseased tissue orcan be used to prognosticate a diseased state in a tissue. Serialchanges in the concentration of a biomarker in a tissue or a bodilyfluid, e.g., an increase or a decrease in the concentration of saidbiomarker between two or more time points T1 and T2 in said tissue orbodily fluid, can be indicative of a diseased state in a tissue or canbe used to assess the metabolic activity of normal or diseased tissue orcan be used to prognosticate a diseased state in a tissue. Biomarkerscan also be used to monitor response to a therapeutic intervention. Abiomarker can be measured by obtaining tissue samples such as pieces ofa mucosal membrane or by obtaining samples of a bodily fluid.

The term “biomarker test” includes a test that assesses (quantitativelyor qualitatively) the concentration (amount), presence and/or serialchanges in the concentration of a biomarker in a tissue or a bodilyfluid. Bodily fluid includes saliva, sputum, nasal secretions, sweat,urine, blood, plasma, serum, synovial fluid, ascites, peritoneal fluid,fluid in a cyst, fluid in an abscess, cerebrospinal fluid, pleuraleffusions, and pericardial effusions. It is contemplated that the methodmay also be used, for example, on saliva and sweat. The body fluid maybe used as it is, or it may be purified prior to analysis. Thepurification of the bodily fluids may be accomplished using a number ofstandard procedures, including, but not limited to, cartridge adsorptionand elution, molecular sieve chromatography, dialysis, ion exchange,alumina chromarography, hydroxyapatite chromatography, and combinationsthereof.

The term “imaging test” includes, but is not limited to, x-ray basedtechniques, for example, conventional film based x-ray films, digitalx-ray images, single and dual x-ray absorptiometry, and radiographicabsorptiometry; ultrasound including broadband ultrasound attenuationmeasurement and speed of sound measurements; computed tomography;nuclear scintigraphy; SPECT; positron emission tomography and MRI. Oneor more of these imaging tests may be used in the methods describedherein, for example in order to obtain certain information (e.g.,morphological) about one or several tissues such as bone including bonemineral density and curvature of the subchondral bone, cartilageincluding biochemical composition of cartilage, cartilage thickness,cartilage volume, cartilage curvature, marrow including marrowcomposition, synovium including synovial inflammation, lean and fattytissue, and thickness, dimensions and volume of soft and hard tissues.The information, which is preferably expressed as a numerical value, isalso referred to as “imaging descriptors.” The imaging test can beperformed with use of a contrast agent, such as Gd-DTPA in the case ofMRI.

The term “osteoporosis” includes a condition of generalized skeletalfragility in which bone strength is sufficiently weak that fracturesoccur with minimal trauma, often no more than is applied by routinedaily activity. Its primary manifestations are a reduction in bone mass,measured usually as bone mineral density (BMD) and disruption of thenormal skeletal micro-architecture. BMD at any time in adult lifereflects the peak investment in bone mineral at skeletal maturity minusthat which has been subsequently lost. In some cases, low BMD representsfailure to achieve adequate bone mass at skeletal maturity. In othercases, it represents loss of bone.

II. General Overview

The present invention provides, among other things, methods forcombining results obtained from assessment of one or more morphologicalparameters characteristic of a disease with information regardingmetabolic, functional, or physiological parameters to evaluate diseasestatus and to provide a prognosis of disease. In particular, for bonediseases, the present invention combines one or more imaging descriptorswith one or more biomarker tests using a mathematical function in orderto diagnose and/or prognose bone disease. The combining of the twodifferent types of data provides an index that is relevant to thediagnosis of the disease. The imaging descriptors provide informationon, for example, bone mineral density, bone structure, and/or the size,shape and invasiveness of the lesion. The biomarker tests provideinformation on, for example, the rate of growth (or extension) of thelesion. One or more of the imaging descriptors are mathematicallymanipulated (e.g., divided by or multiplied by the measured level of thebiomarker) to derive a diagnostic and/or prognostic index.

The methods described herein may be used, for example, to treat defectsresulting from disease of the cartilage (e.g., osteoarthritis), bonedamage and/or degeneration due to overuse or age. The invention allows,among other things, a health practitioner to evaluate and treat suchdefects. Thus, the methods of the invention provide for improved andmore specific diagnosis of diseases, and provide for fasterdetermination on the treatment type needed as well as treatmentefficacy.

III. Imaging Descriptors

The imaging descriptors provide static assessment of one or moremorphological parameters, such as local thickness for cartilage, BMD orstructure for bone. The imaging descriptors thus provide data on thecharacteristics of the bone, such as bone mass, bone density, localthickness of the bone, structure of the bone, thickness of thecartilage, percent cartilage surface diseased, and the like.

When the imaging descriptors pertain to bone mass, a change in the bonemass can typically be measured by four widely available methods known tothose skilled in the art, including single photon absorptometry, dualphoton absorptometry (DPA), dual-energy x-ray absorptometry (DXA), andquantitative computed tomography quantitative computed tomography (CATscan). These methods are used to measure mineral content in the bone,and some are relatively selective for certain bones or trabecular versuscortical bone. Other methods for obtaining information concerning bonedensity and vitality that may reveal information useful in the diagnosisof various diseases including osteopenia, osteoporosis, and arthritis,include magnetic resonance imaging (MRI) and positron emissiontomographic (PET) techniques. Radiographic absorptometry (RA) is amethod for non-invasive measurement of bone mineral x-rays of the hand.

In another aspect, the imaging test can be performed on any x-ray, forexample a dental x-ray obtained in the mandible or maxilla, x-ray of anextremity such as a hand or a calcaneus, a hip x-ray, a spinal x-ray orany other x-ray to obtain a measurement of bone mineral density. Thex-ray (e.g., mandible, maxilla) can be analyzed to obtain a measurementbone mineral density, and the analysis can be performed with the help ofa calibration phantom or an external standard included on the x-rayimage. The calibration phantom or an external standard can be includedon the x-ray image or can be scanned separately. The x-ray can also beused to assess bone structure, e.g. trabecular thickness and spacing,trabecular connectivity, or cortical thickness. See, for example,International Publications PCT/US01/26913 and PCT/US01/32040,incorporated by reference herein in their entireties.

In another aspect, imaging descriptors providing quantitativeinformation on the bone is derived from the measurement of the bonedensity. The density of the bone can be measured by ultrasound tomeasure broadband ultrasound attenuation values and speed of sound todetermine bone density. However, other types of densitometric systemsare also contemplated. For example, the densitometric bone measuringsystem may use x-rays to measure bone density. An example of an x-raybased densitometric bone measuring system using a pencil beam to measurebone density is described in U.S. Pat. No. 4,811,373.

In another aspect of the invention, imaging descriptors providingquantitative information on the bone is derived from an x-ray image. Inother aspects, the quantitative information is densitometricinformation, for example bone mineral density or density of selectedsoft-tissues or organs. Alternatively, the quantitative information isinformation on the morphology of a structure, for example information onthe two-dimensional arrangement of individual components forming saidstructure or information on the three-dimensional arrangement ofindividual components forming said structure. In any of the methodsdescribed herein, the structure can be bone and the information can be,for example, information on trabecular thickness, trabecular spacingand/or estimates of the two- or three-dimensional architecture of thetrabecular network. Further, in any of the methods described herein,quantitative information can be derived with use of an externalstandard, for example a calibration phantom of known x-ray density.(e.g., a calibration phantom is included with the structure to be imagedon the x-ray image).

In another aspect, the quantitative information derived from the x-rayimage includes one or more parameters relating to the acquisition of thex-ray image (e.g,. x-ray tube voltage, x-ray energy, x-ray tube current,film-focus distance, object-film distance, collimation, focal spot size,spatial resolution of the x-ray system, filter technique, film focusdistance, correction factor(s) or combinations thereof), for instance toimprove the accuracy of the quantitative information.

Preferably the x-ray images include accurate reference markers, forexample calibration phantoms for assessing bone mineral density of anygiven x-ray image. Thus, in certain aspects, methods that allow accuratequantitative assessment of information contained in an x-ray such asx-ray density of an anatomic structure or morphology of an anatomicstructure are used to obtain the imaging descriptors.

An x-ray image can be acquired using well-known techniques from anylocal site. For example, in certain aspects, 2D planar x-ray imagingtechniques are used. 2D planar x-ray imaging is a method that generatesan image by transmitting an x-ray beam through a body or structure ormaterial and by measuring the x-ray attenuation on the other side ofsaid body or said structure or said material. 2D planar x-ray imaging isdistinguishable from cross-sectional imaging techniques such as computedtomography or magnetic resonance imaging. If the x-ray image wascaptured using conventional x-ray film, the x-ray can be digitized usingany suitable scanning device. The digitized x-ray image can then betransmitted over the network, e.g. the internet, into a remote computeror server. It will be readily apparent that x-ray images can also beacquired using digital acquisition techniques, e.g. using phosphorusplate systems or selenium or silicon detector systems, the x-ray imageinformation is already available in digital format. In this case theimage can be transmitted directly over the network, e.g. the Internet,or alternatively, it can be compressed prior to transmission.

Preferably, when an x-ray of an anatomic structure or a non-livingobject is acquired a calibration phantom is included in the field ofview. Any suitable calibration phantom can be used, for example, onethat comprises aluminum or other radio-opaque materials. U.S. Pat. No.5,335,260 describes other calibration phantoms suitable for use inassessing bone mineral density in x-ray images. Examples of othersuitable calibration reference materials can be fluid or fluid-likematerials, for example, one or more chambers filled with varyingconcentrations of calcium chloride or the like.

It will be readily apparent that a calibration phantom can containseveral different areas of different radio-opacity. For example, thecalibration phantom can have a step-like design, whereby changes inlocal thickness of the wedge result in differences in radio-opacity.Stepwedges using material of varying thickness are frequently used inradiology for quality control testing of x-ray beam properties. Byvarying the thickness of the steps, the intensity and spectral contentof the x-ray beam in the projection image can be varied. Stepwedges arecommonly made of aluminum, copper and other convenient and homogeneousmaterials of known x-ray attenuation properties. Stepwedge-like phantomscan also contain calcium phosphate powder or calcium phosphate powder inmolten paraffin.

Alternatively, the calibration reference may be designed such that thechange in radio-opacity is from periphery to center (for example in around, ellipsoid, rectangular of other shaped structure). As notedabove, the calibration reference can also be constructed as plurality ofseparate chambers, for example fluid filled chambers, each including aspecific concentration of a reference fluid (e.g., calcium chloride).

Any shape can be used including, but not limited to, squares, circles,ovals, rectangles, stars, crescents, multiple-sided objects (e.g.,octagons), irregular shapes or the like, so long as their position isknown to correlate with a particular density of the calibration phantom.In preferred embodiments, the calibration phantoms described herein areused in 2D planar x-ray imaging.

Since the density and attenuation of the calibration phantom are bothknown, the calibration phantom provides an external reference formeasuring the density of the anatomic structure or non-living object tobe measured. One of skill in the art will easily recognize otherapplications for use of calibration phantoms in x-ray imaging in view ofthe teachings herein.

Curvature and/or thickness measurements of bone or other tissue can alsobe obtained using any suitable techniques, for example in one direction,two directions, and/or in three dimensions. Non-limiting examples ofimaging techniques suitable for measuring thickness and/or curvature(e.g., of cartilage and/or bone) include the use of x-rays, magneticresonance imaging (MRI), computed tomography scanning (CT, also known ascomputerized axial tomography or CAT), and ultrasound imagingtechniques. (See, also, International Patent Publication WO 02/22014;U.S. Pat. No. 6,373,250 and Vandeberg et al. (2002) Radiology222:430-436).

In certain embodiments, CT or MRI is used to assess tissue, bone and anydefects therein, for example articular cartilage and cartilage lesions,to obtain information on cartilage degeneration and provide morphologicinformation about the area of damage. Specifically, changes such asfissuring, partial or full thickness cartilage loss, and signal changeswithin residual cartilage can be detected using one or more of thesemethods. For discussions of the basic NM principles and techniques, seeMRI Basic Principles and Applications, Second Edition, Mark A. Brown andRichard C. Semelka, Wiley-Liss, Inc. (1999). For a discussion of MRIincluding conventional T1 and T2-weighted spin-echo imaging, gradientrecalled echo (GRE) imaging, magnetization transfer contrast (MTC)imaging, fast spin-echo (FSE) imaging, contrast enhanced imaging, rapidacquisition relaxation enhancement, (RARE) imaging, gradient echoacquisition in the steady state, (GRASS), and driven equilibrium Fouriertransform (DEFT) imaging, to obtain information on cartilage, see WO02/22014. Thus, the measurements may be three-dimensional imagesobtained as described in WO 02/22014. Three-dimensional internal images,or maps, of the cartilage alone or in combination with a movementpattern of the joint can be obtained. In addition, imaging techniquescan be compared over time, for example to provide up to date informationon the size and type of repair material needed.

In another aspect, the thickness of the normal or only mildly diseasedcartilage surrounding one or more cartilage defects is measured. Thisthickness measurement can be obtained at a single point or, preferably,at multiple points, for example 2 point, 4-6 points, 7-10 points, morethan 10 points or over the length of the entire remaining cartilage. Inother embodiments, for example if no cartilage remains, the curvature ofthe articular surface can be measured to design and/or shape the repairmaterial. Further, both the thickness of the remaining cartilage and thecurvature of the articular surface can be measured to determine thepercent of cartilage surface that is diseased.

Alternatively, or in addition to, imaging techniques, measurementsand/or samples can be taken of bone or other tissue intraoperativelyduring arthroscopy or open arthrotomy. At least one, preferably two ormore of these measurements can then be used to provide the data for themorphological parameters.

VI. Biomarker Parameters

Biomarkers of bone disease include any products produced or given offduring normal or abnormal growth and/or death of bone and may be used todetermine normal, healthy conditions as well as disease states.Non-limiting examples of suitable biomarkers include calcium,hydroxyproline, alkaline phosphatase, procollagen Type I and itscleavage products, osteocalcin, and bone collagen peptides that includecrosslinked amino acids. The crosslinked amino acids includepyridinoline, hydroxy lysyl pyridinoline, lysyl pyridinoline,substituted pyridinolines, n-telopeptide, and the peptides that containthese cross-linked amino acids. Current methods used to monitor thepresence, progress of treatment, or disease state for metabolic bonediseases require the measurement of biomarkers of bone metabolism foundin bodily fluids. Examples of these detection methods are shown in U.S.Pat. Nos. 5,283,197; 4,973,666; and 5,140,103.

The biomarkers are detected in any suitable sample, for example, tissueor body fluids including but not limited to urine, blood, serum, plasma,sweat, saliva and synovial fluid. The sample (e.g., fluid or tissue) maybe used as it is, or it may be purified prior to the contacting step.This purification step may be accomplished using a number of standardprocedures, including, but not limited to, cartridge adsorption andelution, molecular sieve chromatography, dialysis, ion exchange, aluminachromatography, hydroxyapatite chromatography, and combinations thereof.

The biomarker(s) to be measured will be selected depending on the typeof disease, particularly the type of bone disease, to be detected. Oneor more biomarkers can be selected so that it is characteristic of thedisease exhibited by the patient. For example, the preferred biomarkerspredictive of bone resorption are n-telopeptides and pyridinole. Thesebiomarkers are indicative of resorption and are present in the bodilyfluids in amounts that are detectable and indicative of resorption bonediseases. Thus, the measurement of these biomarkers can provide anindication of the metabolic bone disease, and can be of use inmonitoring the progress of medical treatment intended to reduce the lossof bone density found in these diseases.

Osteoporosis and osteopenia provide other examples of diseases for whichsuitable biomarkers are known. Examples of biomarkers, which can bedetected separately or together show characteristic changes in thepresence of osteoporosis include: calcium, phosphate, estradiol(follicular, mid-cycle, luteal, or post-menopausal), progesterone(follicular, mid-cycle, luteal, mid-luteal, oral contraceptive, or over60 years), alkaline phosphatase, percent liver-ALP, and totalintestinal-ALP. Typically, after measuring the amount of one or more ofthese biomarkers, a diagnosing clinician compares the measurements to anormal reference range to determine weather a patient has undergone somebone loss.

Biomarkers typically monitored for bone resorption associated withPaget's disease includes hydroxyproline. Paget's disease, a metabolicbone disorder in which bone turnover is greatly increased.Hydroxyproline is an amino acid largely restricted to collagen, is theprincipal structural protein in bone and all other connective tissues,and is excreted in urine. The excretion rate of hydroxyproline is knownto be increased in certain conditions, particularly in Paget's disease.Therefore, urinary hydroxyproline can be used as an amino acid markerfor collagen degradation. U.S. Pat. No. 3,600,132 discloses a processfor the determination of hydroxyproline in body fluids such as serum,urine, lumbar fluid and other intercellular fluids in order to monitordeviations in collagen metabolism. The method correlates hydroxyprolinewith increased collagen anabolism or catabolism associated withpathological conditions such as Paget's disease, Marfan's syndrome,osteogenesis imperfecta, neoplastic growth in collagen tissues and invarious forms of dwarfism. Additionally, bone resorption associated withPaget's disease has been monitored by measuring small peptidescontaining hydroxyproline, which are excreted in the urine followingdegradation of bone collagen.

Other biomarkers of collagen degradation that have been measured by artknown methods includes hydroxylysine and its glycoside derivatives, thecross-linking compound 3-hydroxypyridinium in urine as an index ofcollagen degradation in joint diseases (Wu and Eyre, Biochemistry23:1850 (1984) and Black et al., Annals of the Rheumatic Diseases48:641-644 (1989)) where the peptides from body fluids are hydrolyzedand the presence of individual 3-hydroxypyridinium residues issubsequently detected.

A particularly useful biomarker for determining quantitative boneresorption of type II and type III coliagens involves quantitating in abody fluid the concentration of telopeptides that have a3-hydroxypyridinium cross-link and that are derived from collagendegradation. Telopeptides are cross-linked peptides having sequencesthat are associated with the telopeptide region of, for example, type IIand type III collagens and which may have cross-linked to them a residueor peptide associated with the collagen triple-helical domain. Thetelopeptides can have fewer amino acid residues than the entiretelopeptide domains of type II and type III collagens, and they maycomprise two peptides linked by a pyridinium cross-link and furtherlinked by a pyridinium cross-link to a residue or peptide of thecollagen triple-helical domain. Methods for quantitating in a body fluidthe concentration of telopeptides having a 3-hydroxypyridiniumcross-link derived from bone collagen resorption are known in art. Forexample, GB patent application No. 2,205,643 reports that thedegradation of type III collagen in the body can be quantitativelydetermined by measuring the concentration of an N-terminal telopeptidefrom type III collagen in a body fluid.

Typically, the patient's body fluid is contacted with an immunologicalbinding partner specific to a telopeptide having a 3-hydroxypyridiniumcross-link derived from type II or type III collagen. The body fluid maybe used as is or purified prior to the contacting step. Thispurification step may be accomplished using a number of standardprocedures, including cartridge adsorption and elution, molecular sievechromatography, dialysis, ion exchange, alumina chromatography,hydroxyapatite chromatography, and combinations thereof.

The biomarker telopeptide having a 3-hydroxypyridinium cross-linked canalternatively be quantitated by fluorometric measurement of a body fluidcontaining the biomarker. The fluorometric assay can be conducteddirectly on a body fluid without further purification. However, forcertain body fluids, particularly urine, it is preferred thatpurification of the body fluid be conducted prior to the fluorometricassay. This purification step consists of dialyzing an aliquot of a bodyfluid such as urine against an aqueous solution thereby producingpartially purified peptide fragments retained within the nondiffusate(retentate). The nondiffusate is then lyophilized, dissolved in an ionpairing solution and adsorbed onto an affinity chromatography column.The chromatography column can be washed with a volume of ion pairingsolution and, thereafter, the peptide fragments are eluted from thecolumn with an eluting solution. These purified peptide fragments canthen be hydrolyzed and the hydrolysate resolved chromatographically.Chromatographic resolution may be conducted by either high-performanceliquid chromatography or microbore high performance liquidchromatography.

In another method, the assaying of type I, II and III collagen fragmentsin urine is performed by an inhibition ELISA (enzyme linkedimmunosorbent assay) by metering off a sample of urine and contactingthe sample with a telopeptides and with an antibody, which isimmunoreactive with the telopeptide. The telopeptide can be immobilizedon a solid support, and the antibody is raised against the telopeptide.The combined reagents and sample are incubated, and aperoxidase-conjugated antibody is added. After another incubation, aperoxidase substrate solution is added. Following short finalincubation, the enzyme reaction is stopped, and the absorbance ismeasured at about 450 nm and compared with a standard curve obtainedwith standard solutions by the same procedure.

Other examples of biomarkers which may be sued include osteocalcin, alsoknown as bone GLA protein of BGP, procollagen type I, bone alkalinephosphatase and total alkaline phosphatase. Suitable methods for thedetermination of these markers can be found, for example, in Delmas, P.D., et al., J. Bone Min. Res. 1:333-337 (1986).

Thus, biomarker parameters characteristic of bone disease can bedetected by performing, for example, a quantitative in-vitro diagnostictest on a bodily fluid sample such as blood, urine, saliva or sweat.However, other techniques or methods may also be utilized for obtainingone or more of the biomarker parameter, including, for example, asolid-phase immunoassay technique, a western blotting technique andfluorescent microscopy technique. Various types of assays, such aschemical, enzymatic, and immunochemical assays, may be used to obtainthe biochemical data. Chemical assays may detect, for example,phosphorous and/or calcium. Enzymatic assays may detect, for example,the enzyme action of alkaline phosphatase. Immunochemical assays maydetect biologic compounds, such as pyridinoline, hydroxy lysylpyridinoline, lysyl pyridinoline, substituted pyridinolines, andn-telopeptide, by monoclonal or polyclonal antibodies or specificreceptor proteins.

V. Mathematical Functions

The imaging descriptors are combined with the biomarker parameters usinga mathematical function. The mathematical function can be division,product, sum, logarithmic function, exponential function, and the like.In certain aspects of the invention, one or more of the mathematicalfunctions can be used in combination.

In one aspect of the invention, biochemical assays can be performed tomeasure bone disease and imaging descriptors can be obtained on theaffected area. The results of the biochemical assay can then be dividedby the results from the imaging descriptors to provide a ratio or anindex. By creating the index between the two assays, information aboutthe disease, such as, for example, the progression of the disease can beobtained.

In another aspect, a plurality of biomarkers associated with one or moreselected bone disease can be derived and can be combined with aplurality of imaging descriptors. For example, for osteoporosis,calcium, phosphate, and alkaline phosphatase can be measured, as well asthe thickness of the bone and bone density. The sum of the measuredconcentration of the biomarkers can then be multiplied by the sum of theimaging descriptors to derive an index.

In another aspect, one or more of the selected biomarkers and one ormore of the selected imaging descriptors can be measured over a periodof time and the relationship between the two can then be statisticallyanalyzed. The time period can be seconds, minutes, hours, days, ormonths or any interval therebetween. The biochemical and morphologicaldata can be, for example, obtained at 0, 1, 2, 3, 5, 7, 10, 15, and 24hours.

The rate of change of the morphological parameters can then be comparedwith the rate of change of the biochemical data as a function of time.Alternatively, the biochemical data can be correlated with themorphological data, where the slope of the correlation serves as anindex. The regression statistical analysis of the data can be performedusing the STATA™ v3.1 (Stata Corporation, College Station, Tex.)statistical analysis software program, or any other commerciallyavailable statistical analysis software. The regression analysis can beapplied to every independent parameter measured, for example, biomarkerschosen, bone thickness, BMD, and the like. Statistical significance mayconsist of p values≦0.05, and can be used to determine which of theparameters are aggregately significant in the prediction of progressionof the disease.

In another aspect of the invention, the results can be compared bycombining them mathematically to form a numerical index, such as bytaking their ratio. A ratio formed between the morphological data andthe biochemical data provides an index which is dependent on theprogression of the bone disease and which therefore can be used fordiagnostic purposes for disorders associated with bone diseases.

The methods of the present invention are sensitive and accurate therebyallowing a practitioner to diagnose bone related diseases promptly, andfollow and assess with greater speed and efficiency the treatment ofthese bone diseases with various therapies. For example, when aclinician evaluates the imaging descriptors and the biomarker parametersindividually, the onset of bone related diseases may not be diagnosedpromptly. The clinician normally compares these values for a particularpatient with the range of reference values generated for the same agegroup, and the measured values may fall within this reference range.Thus, the measured test results for the patient may fall within theexpected values for these tests even though the patient has developedearly stages of a bone disease, leading to a misdiagnosis. In contrast,when the results of the two or more independent tests are mathematicallycombined in accordance with the present invention, the resultant valuesprovide for greater diagnostic power. The methods of the presentinvention thus provide for early diagnosis of diseases. Similarly, theprogression of the disease or the effectiveness of the treatment for thedisease can be evaluated with greater speed.

In another aspect of the invention, a database of the ratios is createdfor a particular bone disease. For example, a particular numerical indexresult can be associated with particular patient types. This may be doneby subjecting a range of samples of known disease type to the methodsdescribed above and building up a database of index results. One maythen identify the index of an unknown sample as being typical of aparticular class of sample previously tested.

Thus, the combined analysis of results of the imaging test and theresults of the biomarker test can provide an improved assessment of thepatient's prognosis. For example, the combined analysis of the resultsof the imaging tests and the results from the biomarker tests can helpdetermine the patient's current bone health or degree of osteopenia orosteoporosis or dental disease more accurately. In addition, thecombined analysis provides an estimate for the rate of progression ofosteopenia or osteoporosis or dental disease more accurately.

In another aspect of the invention, the results of an imaging test toassess bone mineral density or bone structure and architecture can becombined with the results of a biomarker test whereby the combination ofthe results can help select the most appropriate form of therapy, e.g.,therapy with an anabolic drug rather than an anti-resorptive drug andvice versa. For example, the combination of the imaging and biomarkerresults can indicate that a patient's osteopenia or osteoporosis ordental disease is largely the result of bone resorption, ananti-resorptive drug such as a bisphosphonate can be prescribed.Alternatively, if the combination of results indicate that a patient'sosteopenia or osteoporosis or dental disease is largely the result oflack of bone formation an anabolic drug, e.g., parathyroid hormone orone of its derivatives, can be prescribed.

VI. Experimental

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperatures, etc.), but some experimental error anddeviation should, of course, be allowed for.

Example 1 Measuring Bone Mineral Density

An image of a body part is obtained using one or more of the followingtechniques: dual x-ray absorptiometry (DXA) (Eastell et al. (1998) NewEngl J. Med 338:736-746); quantitative computed tomography (QCT) (Cann(1988) Radiology 166:509-522); peripheral DXA (pDXA) (Patel et al.(1999) J Clin Densitom 2:397-401); peripheral QCT (pQCT) (Gluer et. al.(1997) Semin Nucl Med 27:229-247); radiographic absorptiometry (RA)(Gluer et. al. (1997) Semin Nucl Med 27:229-247); standard x-raytechnology; and quantitative ultrasound (QUS) (Njeh et al. “QuantitativeUltrasound: Assessment of Osteoporosis and Bone Status” 1999,Martin-Dunitz, London England). The image may be digitized and storedelectronically.

Bone mineral density is then determined by analyzing the image asdescribed in International Publications PCT/US01/26913 andPCT/US01/32040. Data on bone mineral density may be taken at multipletime points from the same patient and may be some or all of the resultsmay be stored in a database.

Example 2 Serum and Urine Bone Biomarkers

Serum and urine biomarkers provide an economical and practical way tomeasure formation and resorption without invasive surgery. Serum andurine bone biomarkers are assayed at baseline and at 1, 3, 6, 12, 18,and 24 months. Serum total ALP (bone formation), ACP andtartrate-resistant ACP (TRAP, bone resorption), calcium, and phosphorusare measured using a Cobas Fara Chemistry Analyzer (Roche Diagnostics,Nutley, N.J.) (Carlson, et al., 1992; Jayo et al., 1995; Jerome, et al.,1994). Serum BGP (bone turnover) assays are performed using anestablished radioimmunoassay. Bone resorption is measured usingFDA-approved N-telopeptide collagen excretion markers (Osteomarke,Ostex, Seattle, Wash.).

Example 3 Combining Imaging and Biomarkers

The value of N-telopeptide at each time point measured in Example 2 isdivided by the bone mineral density value obtained in Example 1. Theratio thus obtained is compared to a database that contains a ratio fromnon-diseased patients. The ratio is then used to determine whether thepatient's osteopenia is predominantly the result of lack of boneformation.

Example 4 Treatment Based on Combining Imaging and Biomarkers

The patient of Example 3 diagnosed as having osteopenia due to the lackof bone formation is treated with parathyroid hormone, an anabolic drug.The imaging and biomarker tests described in Examples 1 and 2 arerepeated, and the results combined according to Example 3. The indexobtained is used to follow the effectiveness of the treatment with thedrug. The differences in the index value at different time points can beused to assess the efficacy of treatment of the patient forosteoporosis.

Thus, novel methods for diagnosing and prognosing diseases, such as boneloss diseases, are disclosed. Although preferred embodiments of thesubject invention have been described in some detail, it is understoodthat obvious variations can be made without departing from the spiritand the scope of the invention as defined by the appended claims.

What is claimed is:
 1. A method for diagnosis of bone disease, themethod comprising: using a mathematical function to relate (i) the levelof one or more biomarkers with (ii) a numerical value relating to one ormore imaging descriptors comprising predetermined features from imagesdefining bone disease characteristics to obtain a test value; andcomparing the test value with a control value, wherein a test valuewhich differs from the control value by a predetermined amount isindicative of bone disease.
 2. The method of claim 1, wherein the one ormore biomarkers are selected from the group consisting of calcium,hydroxyproline, alkaline phosphatase, procollagen Type I and itscleavage products, osteocalcin, bone collagen peptides, pyridinoline,hydroxy lysyl pyridinoline, lysyl pyridinoline, n-telopeptide andcombinations thereof
 3. The method of claim 2, wherein the biomarker iscalcium.
 4. The method of claim 2, wherein the biomarker ishydroxyproline.
 5. The method of claim 2, wherein the biomarker isalkaline phosphatase.
 6. The method of claim 1, wherein the one or moreimaging descriptors are selected from the group consisting of bone mass,bone density, local thickness of the bone, structure of the bone,thickness of the cartilage, percent cartilage surface diseased andcombinations thereof
 7. The method of claim 6, wherein the imagingdescriptor is bone mass.
 8. The method of claim 6, wherein the imagingdescriptor is bone density.
 9. The method of claim 6, wherein theimaging descriptor is thickness of the cartilage.
 10. The method ofclaim 1, wherein the mathematical function is selected from the groupconsisting of division, product, sum, logarithmic function, exponentialfunction, or combinations thereof.
 11. The method of claim 10, whereinthe mathematical function is division.
 12. The method of claim 10,wherein the mathematical function is product.
 13. The method of claim10, wherein the mathematical function is sum.
 14. The method of claim 1,wherein the bone disease is bone loss.
 15. The method of claim 14,wherein bone loss is associated with a condition selected from the groupconsisting of osteporosis, arthritis, Paget's disease, and periodontaldiseases.
 16. The method of claim 15, wherein bone loss is associatedwith osteoporosis or arthritis.
 17. The method of claim 16, wherein boneloss is associated with osteoporosis and the biomarker is selected fromthe group consisting of hydroxyproline, telopeptide, alkalinephosphatase and combinations thereof and the imaging descriptor isselected for the group consisting of local thickness for cartilage, bonemineral density, bone structure and combinations thereof.
 18. The methodof claim 1, wherein the control value is determined using themathematical formula to relate (i) the level of one or more biomarkersin a normal subject with (ii) a numerical value relating to one or moreimaging descriptors comprising predetermined features from imagesdefining bone disease characteristics in a normal subject.
 19. Themethod of claim 1, wherein the level of one or more biomarkers isdetermined by immunological assay.
 20. The method of claim 1, wherein atest value greater than the control value is indicative of bone disease.